In the neonatal rodent spinal cord, the combination of serotonin and NMDA is adequate to activate the central pattern generator (CPG) networks for locomotion, but the cellular and biophysical mechanisms by which these transmitters organize and activate the CPG are poorly understood. We propose that these neuromodulators reconfigure the intrinsic properties of the CPG neurons and the strengths of network synapses to organize the network into a functional "idling" state, so that descending glutamatergic or sensory input can rapidly initiate locomotion. The neonatal mouse spinal cord is an excellent preparation to test these hypotheses: several neuronal candidates for the CPG have been identified using transgenic and anatomical methods to label specific interneuron types. Two identified classes of interneurons are thought to play important roles in the organization of the mouse spinal locomotor CPG: commissural interneurons (CINs) which coordinate left-right movements, and the Hb9 interneurons which may participate in the rhythm-generating component of the CPG. Following a research approach we have pursued for many years in the crustacean stomatogastric ganglion, we propose to study how the intrinsic cellular properties of these neurons shape their activity patterns during fictive locomotion, and how serotonin and NMDA affect those intrinsic properties. Our first aim is at the cellular level: using a combination of whole cell recording and calcium imaging, we will study the intrinsic firing properties of synaptically isolated interneurons, their modulation by serotonin, and its interaction with NMDA. The goal of this aim is to better understand how modulators can alter the neurons'activity to activate the motor pattern. Second, at the biophysical level, we will use voltage clamp methods to identify the ionic currents affected by serotonin and NMDA in CINs and Hb9 interneurons, to understand the biophysical basis for modulatory changes in the neurons'intrinsic firing properties. Third, we will begin to explore the plasticity of the intrinsic properties of these spinal interneurons by studying how they change during postnatal development, during the time the animal learns to walk, and following spinal cord injury. These projects will elucidate some of the cellular and molecular mechanisms that neuromodulators use to shape the locomotor CPG. Spinal cord injury causes loss not only of the rapid activating signals for locomotion, but also of the slower modulatory inputs that enable the network to function at all. To learn how to restore movement after spinal cord injury, we must understand both the modulatory mechanisms that enable the network to function and the rapid activating mechanisms in the locomotor CPG. PUBLIC HEALTH RELEVANCE We hypothesize that serotonin and other modulators modify the firing properties of locomotor network neurons and their synapses to enable the spinal network to produce the commands for locomotion. When these inputs are lost following spinal cord lesions, the network becomes non-functional. An eventual goal of our work is to provide a rational basis for post-injury neuromodulator therapy, to help maintain the locomotor networks in a functional state until regrowth of axons can be accomplished across the lesion.